Pyrethroids constitute a major class of chemical pesticides. They are synthetic analogues of the natural pyrethrins, which are produced in the flowers of the pyrethrum plant (Tanacetum cinerarifolium). Modification of their structure has yielded compounds that retain the intrinsically modest vertebrate toxicity of the natural products but are both more stable and more potent as pesticides. In the thirty years since their introduction they have risen to about 10-20% of insecticide sales worldwide and they are projected to retain substantial market share into the forseeable future. They are now widely used across agricultural production and processing systems in many countries and have caused residue incidents in diverse commodities ranging from cotton and horticulture through to wool.
Residues of pyrethroid pesticides are undesirable contaminants of the environment and a range of commodities. They are undesirable because of the broad target range of the pesticide across invertebrates and their significant toxicity to vertebrates, although they are generally considered to be amongst the safest pesticides to mammals. Areas of particular sensitivity include contamination of soil, irrigation tailwater that is re-cycled, used by irrigators downstream or simply allowed to run off-farm, and residues above permissible levels in horticultural exports. Animal industries also have problems with pesticide-contaminated commodities arising through either their own pesticide use or their reliance on crop products and by-products as fodder. Processing wastes from food processing plants, carpet dye baths and animal dips are also contaminated, sometimes quite heavily, with pesticide residues. Bioremediation strategies are therefore required for eliminating or reducing these pesticide residues.
One proposed bioremediation strategy involves the use of enzymes capable of immobilising or degrading the pesticide residues. Such enzymes may be employed, for example, in bioreactors through which contaminated water could be passed, or in washing solutions after post-harvest disinfestation of fruit, vegetables or animal products to reduce residue levels and withholding times. Suitable enzymes for degrading pesticide residues include OP hydrolases from bacteria, vertebrates and organophosphate (OP) resistant insects. It is desirable that the hydrolytic enzymes degrade the pesticide residues at a rapid rate.
Organophosphate resistance in the sheep blowfly, Lucilia cuprina, is conferred by two different mutations in the gene encoding carboxylesterase E3. The two mutant enzymes differ in their substrate specificities but between them can detoxify two major subtypes of OPs. The E3 gene from L. cuprina was cloned by Newcomb et al. (1997) and, using a combination of DNA sequencing, baculovirus expression and in vitro mutagenesis, these workers identified the two resistance mutations. One is an Asp for Gly substitution at residue 137 in the oxyanion hole region of the active site (Newcomb et al., 1997). The other is a Leu for Trp substitution at residue 251 in the substrate-binding region (Campbell et al., 1998), which results in an increase in malathion carboxylesterase activity as well as the acquisition of OP hydrolase activity.
There is a need for methods and enzymes which can be used for the bioremediation of, for example, soils, foodstuff and water samples contaminated with hydrophobic ester pesticides and toxins.